Cells are the basic structural and functional units of all living organisms. All cells contain cytoplasm surrounded by a plasma, or cell, membrane. Most bacterial and plant cells are enclosed in an outer rigid or semi-rigid cell wall. The cells contain DNA which may be arranged in 1) a nuclear membrane or 2) free in cells lacking a nucleus. While the cell membrane is known to contain naturally occurring ion channels, compounds that are therapeutically advantageous to cells are usually too large to pass through the naturally occurring ion channels. Conventional interventional methods of delivery of compounds into cells have proved difficult in view of the need for the compounds to pass through the cell membrane, cell wall, and nuclear membrane.
Molecular biology has resulted in mapping the genomes of many plants and animals including the mapping of much of the human genome. The potential for advances in the understanding of the genetic basis of diseases is great, as is the potential for the development of therapies to treat such diseases. However, to fully take advantage of these advancements and treatment therapies, methods are needed which will allow for the delivery of desired compounds into the target cells. Accordingly, researchers have undertaken the development of a variety of intracellular delivery methods for inserting genes and other compounds into both plant and animal cells.
For example, calcium phosphate DNA precipitation has been used to deliver genetic material into cells in cell culture. However, one drawback of this method is that the resultant efficiency of transfection (delivery of the genetic material into the cells) and subsequent gene expression has been very low.
Improved transfection has been attained using viral vectors, e.g., adenovirus and retrovirus, but again, difficulties with gene expression have persisted. In addition, substantial concerns regarding antigenicity and the potential of mutant viruses and other possible deleterious effects exist.
Liposomes, manufactured more easily than viral vectors, have shown promise as gene delivery agents. Liposomes have less biological concerns (in that, for example, they are generally non-antigenic) but the efficiency of transfection and gene expression using liposomes has typically been lower than with viruses.
Gene guns, wherein genes are attached to heavy metal particles such as gold, have been used to fire the particles at high speed into cells. However, while gene guns have resulted in gene expression in culture systems, they have not worked well in vivo. Furthermore, the blast of heavy metal particles may cause damage to the cells and may result in the introduction of undesirable foreign materials, e.g. gold particle fragments, into the cells.
Electroporation is another method of delivering genes into cells. In this technique, pulses of electrical energy are applied to cells to create pores or openings to facilitate passage of DNA into the cells. However, electroporation may damage cells, and furthermore has not been shown to be highly effective in vivo.
Various publications disclose the use of lithotripsy shock waves for effecting intracellular gene transfer, as well as the delivery of other compounds, including, for example, Delius, M., et al., “Extracorporeal Shock Waves for Gene Therapy,” Lancet May 27, 1995, 345:1377; Lauer, U., et al., “Towards A New Gene Transfer System: Shock Wave-Mediated DNA Transfer,” J Cell Biochem 1994, 16A:226; Gambihler, S., et al., “Permeabilization of the Plasma Membrane of L1210 Mouse Leukemia Cells Using Lithotripter Shock Waves,” J Membr Biol 1994, 141:267-75; and Mobley, T. B., et al., “Low Energy Lithotripsy with the Lithostar: Treatment Results with 19,962 Genal and Ureteral Calculi,” J Urol 1993, 149:1419-24. Lithotripsy delivers energy in the range of 200-380 bars, and a frequency of 60-120 Hz, but may be as high as 1200 to 1300 bars. The energy and frequency ranges are typically painful to a patient and thus usually require patient sedation. Lithotripsy machines are large and bulky and are typically cost prohibitive. Lauer et al. disclose the delivery of 250 shock waves at 25 kV with a lithotripter to deliver plasmid DNA which expressed hepatitis B virus surface proteins in a HeLa cell suspension. Gambihler et al. (cited above) teach the permeabilization of mouse cells in vitro to deliver dextrans. The lithotripter shock waves are delivered at 25 kV, at a discharge rate of 60/min. Mobley et al. (cited above) disclose the use of lithotripsy to treat renal and ureteral stones. The shock wave pressure was 200 to 380 bar and a generator range of 10 to 29 kV.
Zhang, L., et al., “Ultrasonic direct gene transfer The Establishment of High Efficiency Genetic Transformation System for Tobacco,” Sci Agric. Sin. 1991, 24:83-89 disclose increased gene expression by tobacco using continuous wave ultrasound at 0.5 W/cm2 for 30 minutes. Zhang et al. do not disclose the ultrasound frequency. The high energy level is in a range necessary for poration to result in the cell wall of tobacco plants.
Rubin, et al., 31st Annual Meeting of the American Society of Clinical Oncology, May 20-23, 1995, disclose the injection of hepatic tumors with a plasmid/cationic lipid complex with ultrasound guidance. Ultrasound is disclosed as a visual guide to monitor the injection of the tumors, rather than as an aid to deliver the complex to the liver tumors.
The present invention provides new and/or better methods for delivering compounds, including genetic material, into a cell. The methods of the present invention may provide a significant advantage over prior art methodology, in that enhanced levels of intracellular delivery, and in the case of nucleotides, gene expression, may be achieved. In addition, the process of the present invention may be performed in cell lines which may be otherwise resistant to intracellular delivery and gene expression using other conventional means. These and/or other aspects of the present invention will become apparent from the further discussions herein.